Rice backcross population assessment for iron tolerance through phenotypic and genotypic analyses

Iron toxicity has become a serious issue affecting rice (Oryza sativa L.) production in many irrigated lowland areas. The selection of Fe2+-tolerant rice cultivars under iron toxicity conditions and the identification of molecular markers are good approaches to obtaining tangible results. This study aimed to identify simple sequence repeat (SSR) markers that were associated with iron tolerance traits in a rice backcross population. A total of 117 seedlings from the backcross (BC3F2) of ‘OM6830’/‘AS996’//‘AS996’ were phenotyped at the 4-week-seedling stage at Ton Duc Thang University, Ho Chi Minh City, Vietnam. The rice population was screened in Yoshida nutrient medium supplemented with FeCl2 at a concentration of 150 mg L−1 under greenhouse conditions. Phenotypic analysis was conducted by scoring two parameters, namely, root length and leaf bronzing. Genotypic analysis was carried out on the BC3F2 population by using four markers, i.e., RM6, RM240, RM252, and RM451, for association analysis with iron tolerance. A total of 23 BC3F2 lines were selected on the basis of their higher tolerance (score 1) for Fe2+ compared with the tolerant parental line ‘AS996’. The markers RM6 and RM240 were highly polymorphic and identified different Fe2+-tolerant lines in the BC3F2 population. Among the BC3F3 progeny derived from the selected 23 BC3F2 lines, BC3F3-7 was identified as the most Fe2+-tolerant line. BC3F3-15 was also found to be Fe2+ tolerant. Both lines showed good development capability and provided high yields under stress conditions. These tolerant BC3F3 lines could be further screened with additional SSR markers in future breeding programs aiming to increase rice production in iron-contaminated areas of the Mekong Delta, Vietnam.

Rapid identification of mutations caused by fast neutron bombardment in Medicago truncatula

Background Fast neutron bombardment (FNB) is a very effective approach for mutagenesis and has been widely used in generating mutant libraries in many plant species. The main type of mutations of FNB mutants are deletions of DNA fragments ranging from few base pairs to several hundred kilobases, thus usually leading to the null mutation of genes. Despite its efficiency in mutagenesis, identification of the mutation sites is still challenging in many species. The traditional strategy of positional cloning is very effective in identifying the mutation but time-consuming. With the availability of genome sequences, the array-based comparative genomic hybridization (CGH) method has been developed to detect the mutation sites by comparing the signal intensities of probes between wild-type and mutant plants. Though CGH method is effective in detecting copy number variations (CNVs), the resolution and coverage of CGH probes are not adequate to identify mutations other than CNVs. Results We report a new strategy and pipeline to sensitively identify the mutation sites of FNB mutants by combining deep-coverage whole-genome sequencing (WGS), polymorphism calling, and customized filtering in Medicago truncatula. Initially, we performed a bulked sequencing for a FNB white nodule (wn) mutant and its wild-type like plants derived from a backcross population. Following polymorphism calling and filtering, validation by manual check and Sanger sequencing, we identified that SymCRK is the causative gene of white nodule mutant. We also sequenced an individual FNB mutant yellow leaves 1 (yl1) and wild-type plant. We identified that ETHYLENE-DEPENDENT GRAVITROPISM-DEFICIENT AND YELLOW-GREEN 1 (EGY1) is the candidate gene for M. truncatula yl1 mutant. Conclusion Our results demonstrated that the method reported here is rather robust in identifying the mutation sites for FNB mutants.

Evaluation and Selection of Interspecific Lines of Groundnut (Arachis hypogaea L.) for Resistance to Leaf Spot Disease and for Yield Improvement

Early and late leaf spot are two devastating diseases of peanut (Arachis hypogaea L.) worldwide. The development of a fertile, cross-compatible synthetic amphidiploid, TxAG-6 ([A. batizocoi × (A. cardenasii × A. diogoi)]4x), opened novel opportunities for the introgression of wild alleles for disease and pest resistance into commercial cultivars. Twenty-seven interspecific lines selected from prior evaluation of an advanced backcross population were evaluated for resistance to early and late leaf spot, and for yield in two locations in Ghana in 2006 and 2007. Several interspecific lines had early leaf spot scores significantly lower than the susceptible parent, indicating that resistance to leaf spot had been successfully introgressed and retained after three cycles of backcrossing. Time to appearance of early leaf spot symptoms was less in the introgression lines than in susceptible check cultivars, but the opposite was true for late leaf spot. Selected lines from families 43-08, 43-09, 50-04, and 60-02 had significantly reduced leaf spot scores, while lines from families 43-09, 44-10, and 63-06 had high pod yields. One line combined both resistance to leaf spot and high pod yield, and several other useful lines were also identified. Results suggest that it is possible to break linkage drag for low yield that accompanies resistance. However, results also suggest that resistance was diluted in many of the breeding lines, likely a result of the multigenic nature of resistance. Future QTL analysis may be useful to identify alleles for resistance and allow recombination and pyramiding of resistance alleles while reducing linkage drag.

An advanced lentil backcross population developed from a cross between Lens culinaris × L. ervoides for future disease resistance and genomic studies

Genetically accessible variation to some of the abiotic and biotic stresses are limited in the cultivated lentil (Lens culinaris Medik.) germplasm. Introgression of novel alleles from its wild relative species will be useful for enhancing the genetic improvement of the crop. L. ervoides, one of the wild relatives of lentil, is a proven source of disease resistance for the crop. Here we introduce a lentil advanced backcross (LABC-01) population developed in cultivar ‘CDC Redberry’ background, based on L. ervoides alleles derived from an interspecific recombinant inbred population, LR-59-81. Two-hundred and seventeen individuals of the LABC-01 population at BC2F3:4 generation were screened for the race 0 of anthracnose (Colletotrichum lentis) and stemphylium blight (Stemphylium botryosum) under controlled conditions. The population showed significant variations for both diseases and the transfer of resistance alleles into the elite cultivar was evident. It also segregated for other traits such as days to flowering, seed coat colour, seed coat pattern and flower colour. Overall, we showed that LABC-01 population can be used in breeding programmes worldwide to improve disease resistance and will be available as a valuable genetic resource for future genetic analysis of desired loci introgressed from L. ervoides.

Importance of correcting genomic relationships in single-locus QTL mapping model with an Advanced Backcross population

Advanced Backcross (AB) populations have been widely used to identify and utilize beneficial alleles in various crops such as rice, tomato, wheat and barley. For the development of an AB population, a controlled crossing scheme is used and this controlled crossing along with the selection (both natural and artificial) of agronomically-adapted alleles during the development of AB population may lead to unbalanced allele frequencies in the population. However, it is commonly believed that interval mapping mapping of traits in experimental crosses such as AB populations are immune to the deviations from the expected frequencies under Mendelian segregation. Using two AB populations and simulated data sets as examples, we describe the severity of the problem caused by unbalanced allele frequencies in quantitative trait loci (QTL) mapping and demonstrate how it can be corrected using the linear mixed model having a polygenic effect with the covariance structure (genomic relationship matrix) calculated from molecular markers.

Fine Mapping and Candidate Gene Analysis of the Up Locus Determining Fruit Orientation in Pepper (Capsicum spp.)

Fruit orientation is an important horticultural and domesticated trait, which is controlled by a single semi-dominant gene (up) in pepper. However, the gene underlying up locus has not yet been identified. In this study, the previously detected major QTL UP12.1 was firstly verified using an intraspecific backcross population (n=225) stem from the cross of BB3 (C. annuum) and its wild relative Chiltepin (C. annuum var. glabriusculum) using BB3 as the recurrent parent. Then, a large BC1F2 population (n=1827) was used for recombinant screening to delimit the up locus into an interval with ~169.51 kb in length. Sequence comparison and expression analysis suggested that Capana12g000958, encoding a developmentally regulated G-protein 2, was the most likely candidate gene for up. The findings of this study will form a basis for gene isolation and reveal of genetic mechanism underlying the fruit orientation domestication in pepper.

Major QTLs, qARO1 and qARO9, Additively Regulate Adaxial Leaf Rolling in Rice

Moderate leaf rolling is considered optimal for the ideal plant type in rice (Oryza sativa L.), as it improves photosynthetic efficiency and, consequently, grain yield. Determining the genetic basis of leaf rolling via the identification of quantitative trait loci (QTLs) could facilitate the development of high-yielding varieties. In this study, we identified three stable rice QTLs, qARO1, qARO5, and qARO9, which control adaxial leaf rolling in a recombinant inbred line (RIL) population derived from a cross between Tong 88-7 (T887) and Milyang 23 (M23), using high-density SNP markers. These QTLs controlled the rolling phenotype of both the flag leaf (FL) and secondary leaf (SL), and different allelic combinations of these QTLs led to a wide variation in the degree of leaf rolling. Additive gene actions of qARO1 and qARO9 on leaf rolling were observed in a backcross population. In addition, qARO1 (markers: 01id4854718 and 01asp4916781) and qARO9 (markers: 09id19650402 and 09id19740436) were successfully fine-mapped to approximately 60- and 90-kb intervals on chromosomes 1 and 9, respectively. Histological analysis of near-isogenic lines (NILs) revealed that qARO1 influences leaf thickness across the small vein, and qARO9 affects leaf thickness in the entire leaf and bulliform cell area, thus leading to adaxial leaf rolling. The results of this study advance our understanding of the genetic and molecular bases of adaxial leaf rolling, and this information can be used for the development of rice varieties with the ideal plant type.

Unraveling the Sclerotinia Basal Stalk Rot Resistance Derived From Wild Helianthus argophyllus Using a High-Density Single Nucleotide Polymorphism Linkage Map

Basal stalk rot (BSR), caused by the fungus Sclerotinia sclerotiorum, is a serious disease of sunflower (Helianthus annuus L.) in the humid temperate growing areas of the world. BSR resistance is quantitative and conditioned by multiple genes. Our objective was to dissect the BSR resistance introduced from the wild annual species Helianthus argophyllus using a quantitative trait loci (QTL) mapping approach. An advanced backcross population (AB-QTL) with 134 lines derived from the cross of HA 89 with a H. argophyllus Torr. and Gray accession, PI 494573, was evaluated for BSR resistance in three field and one greenhouse growing seasons of 2017–2019. Highly significant genetic variations (p < 0.001) were observed for BSR disease incidence (DI) in all field screening tests and disease rating and area under the disease progress curve in the greenhouse. The AB-QTL population and its parental lines were genotyped using the genotyping-by-sequencing method. A genetic linkage map spanning 2,045.14 cM was constructed using 3,110 SNP markers mapped on 17 sunflower chromosomes. A total of 21 QTL associated with BSR resistance were detected on 11 chromosomes, each explaining a phenotypic variation ranging from 4.5 to 22.6%. Of the 21 QTL, eight were detected for BSR DI measured in the field, seven were detected for traits measured in the greenhouse, and six were detected from both field and greenhouse tests. Thirteen of the 21 QTL had favorable alleles from the H. argophyllus parent conferring increased BSR resistance.

An advanced lentil backcross population developed from a cross between Lens culinaris × L. ervoides for future disease resistance and genomic studies

Genetically accessible variation to some of the abiotic and biotic stresses are limited in the cultivated lentil (Lens culinaris Medik.) germplasm. Introgression of novel alleles from its wild relative species might be required for enhancing the genetic improvement of the crop. L. ervoides, one of the wild relatives of lentil, is a proven source of disease resistance for the crop. Here we introduce a lentil advanced backcross population (LABC-01) developed in cultivar CDC Redberry background, based on L. ervoides alleles derived from an interspecific recombinant inbred population, LR-59-81. Two-hundred and seventeen individuals of the LABC-01 population at BC2F3:4 generation were screened for the race 0 of anthracnose (Colletotrichum lentis) and stemphylium blight (Stemphylium botryosum) under controlled conditions. The population showed significant variations for both diseases and transfer of resistance alleles into the elite cultivar was evident. It also segregated for other traits such as days to flowering, seed coat colour, seed coat pattern and flower colour. Overall, we showed that LABC-01 population can be used in breeding programs worldwide to improve disease resistance and will be available as a valuable genetic resource for future genetic analysis of desired loci introgressed from L. ervoides.

Biosynthesis of Steroidal Alkaloids Are Coordinately Regulated and Differ Among Tomatoes in the Red-Fruited Clade

Background/rationale: We quantitatively profiled and genotyped two tomato populations representing diversity in the red-fruited clade to address the lack of knowledge regarding the chemical diversity, concentration, and genetic architecture controlling tomato steroidal alkaloids. Methods: We grew 107 genetically diverse fresh market, processing, land-race, and wild tomatoes in multiple environments. Nine steroidal alkaloids were quantified using ultra-high performance liquid chromatography tandem mass spectrometry. The diversity panel and a biparental population segregating for high alpha-tomatine, were genotyped to identify and validate quantitative trait loci (QTL) associated with steroidal alkaloids. Results: Land-races and wild material exhibited higher alkaloid concentrations and more chemical diversity. Average total content of steroidal alkaloids, often dominated by lycoperoside F/G/esculeoside A, ranged from 1.9 to 23.3 mg/100 g fresh weight across accessions. Land-race and wild cherry accessions distinctly clustered based on elevated early or late steroidal alkaloid concentrations. Significant correlations were observed among early and late steroidal alkaloids in a species-dependent manner. A QTL controlling multiple, early steroidal alkaloid pathway intermediates on chromosome 3 was identified by genome wide association (GWAS) and validated in a backcross population. Conclusions: Tomato steroidal alkaloids are diverse in the red-fruited tomato clade and their biosynthesis is regulated in a coordinated manner.